The challenge of standardizing CAR-T cell monitoring: A comparison of two flow-cytometry methods and correlation with qPCR technique.

Chimeric antigen receptor (CAR) T-cell therapy is a breakthrough in hematologic malignancies, such as acute B lymphoblastic leukemia (B-ALL). Monitoring this treatment is recommended, although standardized protocols have not been developed yet. This work compares two flow cytometry monitoring strategies and correlates this technique with qPCR method. CAR-T cells were detected by two different flow-cytometry protocols (A and B) in nine blood samples from one healthy donor and five B-ALL patients treated with Tisagenlecleucel (Kymriah®, USA). HIV-1 viral load allowed CAR detection by qPCR, using samples from seven healthy donors and nine B-ALL patients. CAR detection by protocol A and B did not yield statistically significant differences (1.9% vs. 11.8% CD3 + CAR+, p = 0.07). However, protocol B showed a better discrimination of the CD3 + CAR+ population. A strong correlation was observed between protocol B and qPCR (r = 0.7, p < 0.0001). CD3 + CAR+ cells were detected by flow cytometry only when HIV-1 viral load was above 104 copies/mL. In conclusion, protocol B was the most specific flow-cytometry procedure for the identification of CAR-T cells and showed a high correlation with qPCR. Further efforts are needed to achieve a standardized monitoring approach.

Prevalence of Chagas disease in Latin American pregnant women in Madrid, Spain: A multicentre cross-sectional study from 2011 to 2016.

OBJECTIVE: The aim of this study is to assess Trypanosoma cruzi infection prevalence among pregnant migrants living in Madrid according to the country of origin and to assess screening coverage in this at-risk population. METHODS: Retrospective multicentre cross-sectional study conducted from January 2011 to December 2016 in eight Madrid hospitals. Each hospital reviewed their microbiology data records to assess the screening coverage and serological diagnosis in all pregnant women coming from endemic areas. RESULTS: From 2011 to 2016, 149,470 deliveries were attended at the eight hospitals, and 11,048 pregnant women were screened for Chagas disease. Most cases (93.5%) were in women from Bolivia, who also showed the highest prevalence (12.4%, 95% confidence interval: 9.9-15.0). Pooled prevalence amongst the screened women was 2.9% (95% CI: 1.8-4.1). Chagas disease screening coverage varied greatly between centres, with a pooled mean coverage of 47% (95% CI: 37%-57%; 73% [95% CI: 63%-82%] for those centres with universal screening vs. 10% [95% CI: 6%-15%] for those with a selective screening approach; p < 0.001). CONCLUSION: Our study provides useful data for policy makers and epidemiologists in a non-endemic area without congenital Chagas screening programmes.

The algorithm used for the interpretation of doravirine transmitted drug resistance strongly influences clinical practice and guideline recommendations.

OBJECTIVES: We report the results of the reverse transcriptase (RT)/protease (PR) transmitted drug resistance (TDR) prevalence study in 2018, focusing on doravirine resistance-associated mutations and the differences observed when Stanford or French National Agency for AIDS Research (ANRS)/Spanish Network of AIDS Research (RIS)/IAS-USA resistance interpretation algorithms are used to describe clinically relevant resistance. METHODS: We used the WHO 2009 list to investigate the prevalence of NNRTI, NRTI and PI TDR, in treatment-naive HIV-1-infected patients, adding mutations E138A/G/K/Q/R, V106I, V108I, V179L, G190Q, H221Y, F227C/L/V, M230IDR, L234I, P236L and Y318F in RT. The prevalence of doravirine resistance-associated mutations, as described by Soulie et al. in 2019, was evaluated. Clinically relevant TDR was investigated using the latest versions of ANRS, RIS, IAS-USA and Stanford algorithms. RESULTS: NNRTI mutations were detected in 82 of 606 (13.5%) patients. We found 18 patients (3.0%) with NRTI mutations and 5 patients (0.8%) with PI mutations. We detected 11 patients harbouring doravirine resistance-associated mutations (prevalence of 1.8%). Furthermore, we observed important differences in clinically relevant resistance to doravirine when ANRS/RIS (0.7%), IAS-USA (0.5%) or Stanford algorithms (5.0%) were used. V106I, which was detected in 3.8% of the patients, was the main mutation driving these differences. V106I detection was not associated with any of the clinical, demographic or virological characteristics of the patients. CONCLUSIONS: The prevalence of NRTI and PI TDR remains constant in Spain. Doravirine TDR is very infrequent by RIS/ANRS/IAS-USA algorithms, in contrast with results using the Stanford algorithm. Further genotype-phenotype studies are necessary to elucidate the role of V106I in doravirine resistance.

High efficacy of resistance-guided retreatment of HCV patients failing NS5A inhibitors in the real world.

BACKGROUND & AIMS: Most hepatitis C virus (HCV)-infected patients failing NS5A inhibitors develop resistance-associated substitutions (RASs). Here we report the use of resistance-guided retreatment of patients who failed prior NS5A inhibitor-containing regimens in the GEHEP-004 cohort. This is the largest direct-acting antiviral (DAA)-resistance cohort study conducted in Spain. We aim to provide indications on how to use resistance information in settings where sofosbuvir/velpatasvir/voxilaprevir may not be available. METHODS: GEHEP-004 is a prospective multicenter cohort enrolling HCV-infected patients treated with interferon (IFN)-free DAA regimens. Prior to retreatment, population-based sequencing of HCV NS3, NS5A and NS5B genes was performed. After receiving a comprehensive resistance interpretation report, the retreatment regimen was chosen and the sustained virological response (SVR) at 12 weeks after treatment completion (SVR12) was recorded. RESULTS: A total of 342 patients experiencing virological failure after treatment with sofosbuvir/ledipasvir±ribavirin (54%), sofosbuvir/daclatasvir±ribavirin (23%), or paritaprevir-ritonavir/ombitasvir±dasabuvir±ribavirin (20%) were studied. After a resistance report, 186 patients were retreated. An SVR12 was achieved for 88.1% of the patients who failed after sofosbuvir/ledipasvir±ribavirin, 83.3% of the patients who failed after sofosbuvir/daclatasvir±ribavirin, 93.7% of the patients who failed after paritaprevir-ritonavir+ombitasvir±dasabuvir±ribavirin. CONCLUSIONS: In our study, we show how resistance-guided retreatment in conjunction with an interpreted report allows patients to achieve SVR rates close to 90%. We hypothesize that SVR rates may even be improved if resistance data are discussed between experienced virologists and treating clinicians. We believe that our data may be relevant for countries where the access to new DAA combination regimens is limited. LAY SUMMARY: Hepatitis C infection can be cured with currently available antiviral agents. Only a small proportion of patients experience treatment failure, however, in absolute numbers, a high number of patients may require retreatment. Highly effective combinations of antivirals are also available for retreatment. However, these antivirals might not be available in resource-limited settings. Herein, we show how, by analyzing the cause of resistance, retreatment efficacy with old drugs can get very close to the efficacy of new drug combinations.

Surveillance of transmitted drug resistance to integrase inhibitors in Spain: implications for clinical practice.

BACKGROUND: Integrase strand-transfer inhibitors (INSTIs) constitute at present one of the pillars of first-line ART. OBJECTIVES: To study the prevalence of and the trend in transmitted drug resistance (TDR) to INSTIs in ART-naive patients in Spain. METHODS: During the period 2012-17, 1109 patients from CoRIS were analysed. The Stanford algorithm v8.7 was used to evaluate TDR and transmission of clinically relevant resistance. To describe individual mutations/polymorphisms, the most recent IAS list (for INSTIs) and the 2009 WHO list update (for the backbone NRTIs used in combination with INSTIs in first-line treatment) were used. RESULTS: Clinically relevant resistance to the INSTI class was 0.2%: T66I, 0.1%, resistance to elvitegravir and intermediate resistance to raltegravir; and G163K, 0.1%, intermediate resistance to raltegravir and elvitegravir. No clinical resistance to dolutegravir or bictegravir was observed. The prevalence of INSTI TDR following the IAS-USA INSTI mutation list was 2.6%, with no trend towards changes in the prevalence throughout the study period. The overall prevalence of NRTI WHO mutations was 4.3%, whereas clinically relevant resistance to tenofovir, abacavir and emtricitabine/lamivudine was 1.7%, 1.9% and 0.7%, respectively. CONCLUSIONS: Given the low prevalence of clinically relevant resistance to INSTIs and first-line NRTIs in Spain, it is very unlikely that a newly diagnosed patient will present with clinical resistance to a first-line INSTI-based regimen. These patients may not benefit from INSTI and NRTI baseline resistance testing.

Prospective comparative multi-centre study on imported Plasmodium ovale wallikeri and Plasmodium ovale curtisi infections.

BACKGROUND: Few previous retrospective studies suggest that Plasmodium ovale wallikeri seems to have a longer latency period and produces deeper thrombocytopaenia than Plasmodium ovale curtisi. Prospective studies were warranted to better assess interspecies differences. METHODS: Patients with imported P. ovale spp. infection diagnosed by thick or thin film, rapid diagnostic test (RDT) or polymerase chain reaction (PCR) were recruited between March 2014 and May 2017. All were confirmed by DNA isolation and classified as P. o. curtisi or P. o. wallikeri using partial sequencing of the ssrRNA gene. Epidemiological, analytical and clinical differences were analysed by statistical methods. RESULTS: A total of 79 samples (35 P. o. curtisi and 44 P. o. wallikeri) were correctly genotyped. Males predominate in wallikeri group (72.7%), whereas were 48.6% in curtisi group. Conversely, 74.3% of curtisi group were from patients of African ethnicity, whilst 52.3% of Caucasians were infected by P. o. wallikeri. After performing a multivariate analysis, more thrombocytopaenic patients (p = 0.022), a lower number of platelets (p = 0.015), a higher INR value (p = 0.041), and shorter latency in Caucasians (p = 0.034) were significantly seen in P. o. wallikeri. RDT sensitivity was 26.1% in P. o. curtisi and 42.4% in P. o. wallikeri. Nearly 20% of both species were diagnosed only by PCR. Total bilirubin over 3 mg/dL was found in three wallikeri cases. Two patients with curtisi infection had haemoglobin under 7 g/dL, one of them also with icterus. A wallikeri patient suffered from haemophagocytosis. Chemoprophylaxis failed in 14.8% and 35% of curtisi and wallikeri patients, respectively. All treated patients with various anti-malarials which included artesunate recovered. Diabetes mellitus was described in 5 patients (6.32%), 4 patients of wallikeri group and 1 curtisi. CONCLUSIONS: Imported P. o. wallikeri infection may be more frequent in males and Caucasians. Malaria caused by P. o. wallikeri produces more thrombocytopaenia, a higher INR and shorter latency in Caucasians and suggests a more pathogenic species. Severe cases can be seen in both species. Chemoprophylaxis seems less effective in P. ovale spp. infection than in P. falciparum, but any anti-malarial drug is effective as initial treatment. Diabetes mellitus could be a risk factor for P. ovale spp. infection.

Hepatitis with a multiple etiology in HIV-positive men who have sexual relations with other men.

Acute hepatitis C virus (HCV) is now a major health problem, mainly in men who have sexual relations with other men (MSM). Hepatitis E virus (HEV) causes sporadic cases of acute hepatitis. In this article, we describe two cases of acute hepatitis due to HCV with an interesting clinical progress.

Comparison of imported Plasmodium ovale curtisi and P. ovale wallikeri infections among patients in Spain, 2005-2011.

Sequencing data from Plasmodium ovale genotypes co-circulating in multiple countries support the hypothesis that P. ovale curtisi and P. ovale wallikeri are 2 separate species. We conducted a multicenter, retrospective, comparative study in Spain of 21 patients who had imported P. ovale curtisi infections and 14 who had imported P. ovale wallikeri infections confirmed by PCR and gene sequencing during June 2005-December 2011. The only significant finding was more severe thrombocytopenia among patients with P. ovale wallikeri infection than among those with P. ovale curtisi infection (p = 0.031). However, we also found nonsignificant trends showing that patients with P. ovale wallikeri infection had shorter time from arrival in Spain to onset of symptoms, lower level of albumin, higher median maximum core temperature, and more markers of hemolysis than did those with P. ovale curtisi infection. Larger, prospective studies are needed to confirm these findings.

Molecular surveillance of HIV-1 in Madrid, Spain: a phylogeographic analysis.

The molecular epidemiology of HIV-1 is constantly changing, mainly as a result of human migratory flows and the high adaptive ability of the virus. In recent years, Spain has become one of Europe's main destinations for immigrants and one of the western European countries with the highest rates of HIV-positive patients. Using a phylogeographic approach, we have analyzed the relationship between HIV-1 variants detected in immigrant and native populations of the urban area of Madrid. Our project was based on two coincidental facts. First, resistance tests were extended to naïve and newly diagnosed patients, and second, the Spanish government legislated the provision of legal status to many immigrants. This allowed us to obtain a large data set (n = 2,792) from 11 Madrid hospitals of viral pol sequences from the two populations, and with this unique material, we explored the impact of immigration in the epidemiological trends of HIV-1 variants circulating in the largest Spanish city. The prevalence of infections by non-B HIV-1 variants in the studied cohort was 9%, rising to 25% among native Spanish patients. Multiple transmission events involving different lineages and subsubtypes were observed in all the subtypes and recombinant forms studied. Our results also revealed strong social clustering among the most recent immigrant groups, such as Russians and Romanians, but not in those groups who have lived in Madrid for many years. Additionally, we document for the first time the presence of CRF47_BF and CRF38_BF in Europe, and a new BG recombinant form found in Spaniards and Africans is tentatively proposed. These results suggest that the HIV-1 epidemic will evolve toward a more complex epidemiological landscape.

Imported Plasmodium falciparum malaria in HIV-infected patients: a report of two cases.

As HIV becomes a chronic infection, an increasing number of HIV-infected patients are travelling to malaria-endemic areas. Association of malaria with HIV/AIDS can be clinically severe. Severe falciparum malaria is a medical emergency that is associated with a high mortality, even when treated in an Intensive Care Unit. This article describes two cases of HIV-positive patients, who returned from malaria-endemic areas and presented a parasitaemia > 5% of erythrocytes and clinical signs of severe falciparum malaria, both with > 350 CD4 cell count/µl, absence of chemoprophylaxis and successful response. Factors like drug interactions and the possible implication of anti-malarial therapy bioavailability are all especially interesting in HIV-malaria co-infections.

Urinary Tract Infections in Hospitalized COVID-19 Patients, What's Up, Doc?

The SARS-CoV-2 pandemic might have increased the risks of healthcare-associated infections (HAIs); however, several studies of HAI such as urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs) have shown contradictory results. The aim of this study is to assess the clinical features of UTIs and bacterial isolates from urine samples of hospitalized COVID-19 patients. We conducted a retrospective observational study including 87 COVID-19 patients with UTIs admitted to our centre. Bacterial UTIs presented were 87: 9 (10.3%) community-acquired UTIs (coinfection group) and 78 (89.6%) hospital-acquired UTIs (superinfection group). In the coinfection group, the most frequent type was non-CAUTI with 5 (55.5%) patients; however, the most frequent UTI in the superinfection group was CAUTI, with 53 (67.9%) patients. The median number of days of hospitalization in coinfected patients was lower than superinfection patients: 13 (IQR 11, 23) vs. 34 days (IQR 23, 47) p < 0.006. All UTI patients admitted to ICU, 38 (43.7%), belonged to the superinfection group. The mortality rate was 26.4% (23/87), 22/23 in the superinfection group. The most common microorganisms were E. coli 27 (28.4%), E. faecalis 25 (26.3%) and E. faecium 20 (21.1%). There was an increased incidence of E. faecalis and E. faecium in UTIs as well as hospital-acquired UTIs. This can be related to urethral catheterization during hospitalization, UCI admissions and the number of days of hospitalization.

Characteristics of patients with suspected COVID-19 pneumonia and repeatedly negative RT-PCR.

OBJECTIVES: Challenges remain and there are still a sufficient number of cases with epidemiological, clinical features and radiological data suggestive of COVID-19 pneumonia that persist negative in their RT-PCR results. The aim of the study was to define the distinguishing characteristics between patients developing a serological response to SARS-CoV-2 and those who did not. METHODS: RT-PCR tests used were TaqPath 2019-nCoV Assay Kit v1 (ORF-1ab, N and S genes) from Thermo Fisher Diagnostics and SARS-COV-2 Kit (N and E genes) from Vircell. Serological response was tested using the rapid SARS-CoV2 IgG/IgM Test Cassette from T and D Diagnostics Canada and CMC Medical Devices and Drugs, S.L, CE. RESULTS: In this cross-sectional study, we included a cohort of 52 patients recruited from 31 March 2020 to 23 April 2020. Patients with positive serology had an older average age (73.29) compared to those who were negative (54.82) (P<0.05). Sat02 in 27 of 34 patients with positive serology were below 94% (P<0.05). There was a frequency of 1.5% negative SARS-CoV-2 RT-PCRs during the study period concurring with 36.7% of positivity. CONCLUSION: Clinical features and other biomarkers in a context of a positive serology can be considered crucial for diagnosis.

Characteristics of Zika virus infection among international travelers: A prospective study from a Spanish referral unit.

BACKGROUND: From the first Zika virus (ZIKV) description, it has progressively widespread worldwide. We analyzed demographic, clinical, microbiologic and travel-related characteristic from returned patients from a ZIKV endemic country in a referral Tropical Medicine Unit. METHOD: A prospective cohort study performed in a Spanish referral center with the aim of determining the significant factors associated with confirmed Zika virus (ZIKV) infection. RESULTS: 817 patients, (56% women, median age 36 [IQR, Interquartile Range: 32-42]) were enrolled. Most had returned from Latin America (n = 486; 59.4%), travelled for tourism (n = 404; 49.4%) and stayed a median of 18 days (IQR: 10-30). 602 (73.6%) presented symptoms, but only 25 (4%) were finally diagnosed with confirmed ZIKV infection (including two pregnant women, without adverse fetal outcomes), 88% (n:22) presented with fever and 92% (n:23) with rash. 56% (n:14) arthralgia and/or myalgia and 28% (n:7) conjunctivitis. The presence of conjunctivitis, fever and rash were associated with an 8.9 (95% CI: 2.2-34.9), 6.4 (95% CI: 1.2-33.3) and 72.3 (95% CI: 9.2-563.5) times greater probability of confirmed ZIKV infection, respectively. CONCLUSION: Travel characteristics and clinical presentation may help clinicians to optimize requests for microbiological testing. Diagnosis of arboviriasis in travellers arriving form endemic areas remains a challenge for clinicians, but must be detected for the possible transmission outside endemic areas, where the vector is present.

GEHEP 010 study: Prevalence and distribution of hepatitis B virus genotypes in Spain (2000-2016).

OBJECTIVE: To study the prevalence and distribution of HBV genotypes in Spain for the period 2000-2016. METHODS: Retrospective study recruiting 2559 patients from 17 hospitals. Distribution of HBV genotypes, as well as sex, age, geographical origin, mode of transmission, HDV-, HIV- and/or HCV-coinfection, and treatment were recorded. RESULTS: 1924 chronically HBV native Spanish patients have been recruited. Median age was 54 years (IQR: 41-62), 69.6% male, 6.3% HIV-coinfected, 3.1% were HCV-coinfected, 1.7% HDV-co/superinfected. Genotype distribution was: 55.9% D, 33.5% A, 5.6% F, 0.8% G, and 1.9% other genotypes (E, B, H and C). HBV genotype A was closely associated with male sex, sexual transmission, and HIV-coinfection. In contrast, HBV genotype D was associated with female sex and vertical transmission. Different patterns of genotype distribution and diversity were found between different geographical regions. In addition, HBV epidemiological patterns are evolving in Spain, mainly because of immigration. Finally, similar overall rates of treatment success across all HBV genotypes were found. CONCLUSIONS: We present here the most recent data on molecular epidemiology of HBV in Spain (GEHEP010 Study). This study confirms that the HBV genotype distribution in Spain varies based on age, sex, origin, HIV-coinfection, geographical regions and epidemiological groups.

Prevalence of resistance associated substitutions and efficacy of baseline resistance-guided chronic hepatitis C treatment in Spain from the GEHEP-004 cohort.

Treatment guidelines differ in their recommendation to determine baseline resistance associated substitutions (RAS) before starting a first-line treatment with direct-acting antivirals (DAAs). Here we analyze the efficacy of DAA treatment with baseline RAS information. We conducted a prospective study involving 23 centers collaborating in the GEHEP-004 DAA resistance cohort. Baseline NS5A and NS3 RASs were studied by Sanger sequencing. After issuing a comprehensive resistance report, the treating physician decided the therapy, duration and ribavirin use. Sustained virological response (SVR12) data are available in 275 patients. Baseline NS5A RAS prevalence was between 4.3% and 26.8% according to genotype, and NS3 RASs prevalence (GT1a) was 6.3%. Overall, SVR12 was 97.8%. Amongst HCV-GT1a patients, 75.0% had >800,000 IU/ml and most of those that started grazoprevir/elbasvir were treated for 12 weeks. In genotype 3, NS5A Y93H was detected in 9 patients. 42.8% of the HCV-GT3 patients that started sofosbuvir/velpatasvir included ribavirin, although only 14.7% carried Y93H. The efficacy of baseline resistance-guided treatment in our cohort has been high across the most prevalent HCV genotypes in Spain. The duration of the grazoprevir/elbasvir treatment adhered mostly to AASLD/IDSA recommendations. In cirrhotic patients infected with GT-3 there has been a high use of ribavirin.

Detection of toxigenic Clostridium difficile in paediatric patients. / Detección de Clostridium difficile toxigénico en pediatría.

INTRODUCTION: Our main objective was a revision of clinical, microbiological and epidemiological results of Clostridium difficile-associated infection in paediatric patients (2010-2015). We compared the diagnoses performed by detection of toxins in feces and those performed by real-time PCR. METHODS: This retrospective study included 82 paediatric patients. Detection of toxigenic C. difficile was performed sequentially, in diarrheal feces and under clinical request. RESULTS: A total of 39% of the patients were attended at Haematology-oncology Unit and >50% of them had previously received cephalosporins. Fever associated with diarrhea was more frequent in the group of toxin detection, whereas not receiving specific antibiotic treatment was more frequent in the group of positive PCR, without statistically significant differences. CONCLUSIONS: We highlight the presence of C. difficile infection in children under 2years old. A diagnostic testing in selected paediatric patients would be advisable when there is clinical suspicion of infection.